The overarching goal of this competing renewal application (P01-DK58398-11) continues to be the development of technologies that lead to new methods for studying, detecting, and treating type 2 diabetes, and their integration with hypothesis-driven diabetes research projects. The program involves collaboration of two major metabolic research centers at Duke University Medical Center and the University of Texas Southwestern Medical Center, Dallas. In the past funding cycle, our team's most compelling advances in technology development have centered on comprehensive tools for metabolic analysis, including NMR-based methods for measurement of metabolic flux and mass spectrometry-based methods for static profiling of intermediary metabolites. A key goal of the program in moving forward is to apply these tools in an integrated fashion to a diverse array of animal models and human subjects to gain a more comprehensive view of metabolic perturbations associated with development of type 2 diabetes than has heretofore been possible. The four projects and three cores of the program are organized around three core hypotheses: 1) Major complications of over-nutrition such as insulin resistance and glucose intolerance are the result of overload of normally functioning mitochondrial pathways rather than intrinsic deficiencies in mitochondrial metabolism; 2) In addition to lipids, branched-chain amino acids and related metabolites play an important role in causation of mitochondrial dysfunction and loss of insulin sensitivity; 3) Mitochondrial flexibility in oxidative and anaplerotic pathways is impaired in insuin resistant liver via constitutive activation of mTORCI and substrate overload. A distinguishing feature of the application is the translation of new understanding and hypotheses developed in cell and animal models in Projects 1, 2, and 3 to human subjects via the studies proposed in Project 4. Through this work, we hope to derive the most complete understanding to date of changes in metabolism within major organs and tissues during development of insulin resistance, type 2 diabetes, and related disorders, leading to novel therapeutic targets and new diagnostic tests for metabolic diseases that cripple modern society.